This invention relates to direct nucleic acid detection, particularly DNA and RNA in low copy number, without a requirement for amplification of the DNA or RNA molecules. The method is directly quantitative, utilizing capillary electrophoresis and laser-induced fluorescence to detect a target DNA-DNA or RNA-DNA probe hybrid band. The invention also relates to analysis of deletion and point mutations.
The quantitation of RNA and DNA derived from infectious agents or from cellular sources is important in the diagnosis and monitoring of a range of disease states. For example, the HIV viral load detected in serum of AIDS patients correlates to high concentrations of virus in the lymph nodes and has predictive value in assessing progression of the disease to advanced stages (see, e.g., Ho, et al., Nature, 373:123 (1995); Mellors, et al., Ann. Intern. Med., 123:573 (1996)). Viral titers in serum are also correlated with disease progression for other viruses such as hepatitis C virus (HCV), nonA nonB hepatitis virus other than HCV, and atypical lentiviruses. The ability to quantify the copy number of DNA (or RNA) may also be useful in the diagnosis and prognostic evaluation of certain cancers. It is increasingly understood that all cancers are multi-step genetic diseases, and a number of genetic defects have been identified, e.g., in pancreatic cancer. Early stage detection of the mutated sequences can be an important tool in a treatment arsenal, but requires quantitation of DNA (or RNA) in low copy number.
The current methods for detecting RNA and DNA quantitation in low copy number are divided into two categories; (i) those that result in the amplification of the target sequence, and (ii) those that result in amplification of a signal sequence. Amplification of the target or signal sequence increases its numbers exponentially but the final result depends upon a large number of reactions that must occur in correct sequence. The coefficients of variability (CV) may often exceed 20% or more, so that the result obtained is unreliable, and does not correlate with, e.g., the stage of disease. The coefficient of variability (CV) is defined as the standard deviation of the values obtained divided by the mean. In any detection technique, a coefficient of variability (CV) of less than 15% is the accepted standard of accuracy.
Additionally, direct measurement of RNA in low copy number in a native sample, even where adequate detection sensitivities can be achieved, is thwarted by the inherent instability of RNA-DNA duplexes. Increasing the length of the hybridized target has been found to increase both sensitivity and stability of the hybrid, but the additional nucleotide sequence combinations increase the chance of nonspecific hybridizing to fragments of host nucleic acids or partial hybridization to nonselected regions of the subject genome, thereby contributing to a falsely inflated positive value. Most of the improvements to date in low RNA copy number quantitation represent attempts to better control the multiple molecular events involved in signal or target amplification strategies.
Three main amplification systems currently available include branched chain signal amplification (bDNA), polymerase chain reaction (PCR) or in the case of a RNA target, reverse transscriptase polymerase chain reaction (RT-PCR), and nucleic acid sequence-based amplification (NASBA). When detection of target RNA is the object, bDNA and RT-PCR involve a first reaction step that converts the system from an RNA target to a DNA target. bDNA involves an isothermal two-step hybridization approach.
An initial probe hybridizing with a complementary probe contains a plurality of noncomplementary sites capable of hybridizing to further DNA strands, which in turn may hybridize sites noncomplementary to the probe sequence. As repeated layers of hybridization occur, a branched DNA structure of extreme complexity is created. The last to be annealed strand in the branched structure carries a reporter. The original DNA target molecule thus gives rise to an amplification of the signal generating capability of the system. A full explanation and description of the bDNA technique is set forth in Fultz, et al., "Quantitation of plasma HIV-I RNA using an ultra sensitive branched DNA (bDNA) assay", in Program and Abstracts of the 2nd National Conference on Human Retroviruses (1995), and product literature, L-6170 Rev. 5.0 for the Quantiplex.TM. HIV-RNA Assay (Chiron Corporation).
In PCR, selected primers are used to define the left and right ends of the target sequence. In RT-PCR, a cDNA is generated from the RNA template, and then an ordinary PCR amplification ensues utilizing left and right primers. Each successive round of synthesis and thermal denaturation causes an exponential increase in the number of progeny strands generated in the system. After the amplification is complete, a probe having a complementary sequence to some portion of the amplicon and carrying a reporter can be used for detecting the amplified target.
In both RT-PCR and bDNA, the original RNA target can theoretically be dispensed with, without impairing the sensitivity of the test, once the conversion to a DNA system has occurred. These methods effectively circumvent the inherent lability of the RNA target or its RNA-DNA duplex hybrid.
PCR, RT-PCR and bDNA share many of the same deficiencies. The systems rely upon the integrity of a large number of successive hybridization events. If an early hybridization event fails for any of a number of reasons such as structural (steric) hindrance, uncorrected mismatch, binding of a defective enzyme molecule, etc., the final number of copies, and therefore, the intensity of the signal will be ablated. These random occurrences help to account for the great sensitivity of the assays coupled with a widely variable CV. Commercial assays normalize variability by co-amplification of an internal standard. To control for variability, an internal standard must be amplified under the identical conditions as the target, yet must be differentiated from the target, an almost impossible task. Introducing an internal standard, however, changes the PCR reaction kinetics itself. Additionally, RT-PCR, while showing some efficacy, is very labor intensive, and not practical under normal clinical laboratory conditions. Furthermore, the use of these systems for mutation analysis is especially problematic because the systems arbitrarily introduce new mutations and routinely incorporate incorrect bases, thus, giving a false positive rate.
NASBA is an isothermal assay which uses a combination of three enzymes and flanking primers to generate multiple RNA copies of original RNA/DNA targets. Each of these serves as a new template for transcription and DNA synthesis steps. The process is initiated upon annealing of two primers, one of which contains a phage promoter, which in the ensuing cDNA provides a point of initiation for transcription. Unlike PCR where the numbers of actual cycles of amplification are nominally controlled by the number of temperature cycles, there is much less control in NASBA. The technique suffers from a lack of uniformity between different target sequences, and in the same target sequence from one run to another. The commercial form of the assay employs three internal calibrators, which are co-amplified with the target sequence.
Three techniques, bDNA, NASBA and RT-PCR described herein were recently compared in a study by Coste, et al., J. Med. Viro., 50: 293 (1996). bDNA was found to be most reproducible with CVs ranging from 6-35%. Better results were achieved at high copy number, 12.4% vs. 31% for low copy number. However, sensitivity was only 68% with a lower level of detection at, e.g., 4000 HIV equivalents. NASBA was the least reliable test with CVs ranging from 13-62%, with CV averages of 20.7% for high copy number and 41.8% for low copy number. Sensitivity was 100% with a lower level of detection at, e.g., 2600 HIV equivalents. RT-PCR had a sensitivity of 93%, but a mean CV of 43%.
For detection of mutations, single strand conformational polymorphism (SSCP) is the most common technique used to evaluate small mutations in DNA, usually a single base change. This technique is based on the premise that DNA fragments varying by a single base pair will have altered migration patterns on gel electrophoresis. The great benefit of SSCP is that unknown single nucleotide mutations (SNPs) can be detected by this methodology.
To perform SSCP, the target sequence of DNA is simultaneously amplified by PCR and labeled with radioactivity. The PCR product is then heated to disassociate the strands and electrophoresed on nondenaturing polyacrylamide gels. Control DNA, without any mutations, is electrophoresed to determine the migration time. If the sample DNA has the same nucleotide sequence as the control DNA, a single band is present. However, if a mutation is present two bands, with differing migration times will be present.
Currently, SSCP relies almost exclusively on PCR. As noted hereinbefore, PCR utilizes exponential amplification, with the gene concentration after such amplification being determined easily and reliably by standard methods. However, the initial gene concentration present in a given sample, can not be determined by routine PCR. Methods involving internal standards and standardized visualization have been employed. Unfortunately, these methods have rarely been validated, and reported CV's range from 18-97%, making these PCR-based methods of limited value in clinical and diagnostic settings.
The other major limitation of PCR, particularly in the context of mutation detection, is the rate of error incorporation. The thermostable DNA polymerase derived from Pyrococcus furiosus (Pfu) is reported to have the lowest average error rate. This enzyme makes an error (i.e., introduces a mutation) every 6.5.times.10.sup.7 base pairs synthesized. Assuming a single copy gene, a 300 base pair product and 40 cycles of amplification, this enzyme can be expected to introduce approximately two mutations in every PCR reaction. Standard sequencing techniques require 8 PCR reactions to sequence a 300 bp fragment. Using Pfu polymerase, at least 16 mutations will be introduced when sequencing this fragment. Additionally, since the coding (cDNA) region of most genes is greater than 2000 bp, a minimum of 48 PCRs, (with 96 new mutations introduced) is required to sequence a single gene. This rate of mutation incorporation is unacceptably high, as clinically significant point mutations are usually present in fractions at or below 1 in 10.sup.6.
This rate of mutation incorporation is especially problematic in evaluating solid tumors. Unlike hematologic malignancies, where clonal expansion, or where a single cancerous cell multiplies to make billions of identical cancer cells, the mutation spectrum in solid tumors is heterogenous and the genetic make-up of individual cells within the tumor is different. At least six different mutations are expected per solid tumor and an individual cell will have some, all or none of the mutations. An additional problem when evaluating clinical tumor samples is the difficulty in obtaining only tumor cells in a given sample. Biopsy samples may contain normal tissue, immunologic infiltrates and precursor lesions as well as the tumor itself. The presence and frequency of these mutations within a tumor will be essential to evaluate when considering the influence of genetic make-up on prognosis, diagnosis and therapeutic decisions.
Despite improvements in the foregoing techniques that may result from optimization of the operating conditions of the assays, and from discovery of reagent combinations that minimize interferences with hybridizations, few rapid, simple, reliable and accurate quantitative methods are available. It is unlikely that variability of existing methods will ever be reduced uniformly to CV values of less than 15%. Priming errors, hybridization interferences and introduction of new mutations cannot be entirely overcome, and misevents occurring early in the sequence of amplification steps have a geometric impact on the result. Even if the level of sensitivity for direct detection of DNA and RNA could be increased by several orders of magnitude over standard UV detection methods, and if the problem of RNA-DNA duplex instability could be solved, fully quantitative direct detection of DNA and RNA sequences, including mutated gene sequences, would provide a viable alternative to current amplification-based methods without introduction of new mutations.